This invention is generally in the field of detection of analytes and biomolecules, and more specifically in the field of multiplex detection and analysis of analytes and biomolecules.
Detection of molecules is an important operation in the biological and medical sciences. Such detection often requires the use of specialized label molecules, amplification of a signal, or both, because many molecules of interest are present in low quantities and do not, by themselves, produce detectable signals. Many labels, labeling systems, and signal amplification techniques have been developed. For example, nucleic acid molecules and sequences have been amplified and/or detected using polymerase chain reaction (PCR), ligase chain reaction (LCR), self-sustained sequence replication (3SR), nucleic acid sequence based amplification (NASBA), strand displacement amplification (SDA), and amplification with Qxcex2 replicase (Birkenmeyer and Mushahwar, J. Virological Methods, 35:117-126 (1991); Landegren, Trends Genetics 9:199-202 (1993)). Proteins have been detected using antibody-based detection systems such as sandwich assays (Mailini and Maysef, xe2x80x9cA sandwich method for enzyme immunoassay. I. Application to rat and human alpha-fetoproteinxe2x80x9d J. Immunol. Methods 8:223-234 (1975)) and enzyme-linked immunosorbent assays (Engvall and Perlmann, xe2x80x9cEnzyme-linked immunosorbent assay (ELISA). Quantitative assay of immunoglobulinxe2x80x9d Immunochemistry 8:871-874 (1971)), and two-dimensional (2-D) gel electrophoresis (Patton, Biotechniques 28: 944-957 (2000)). Although these techniques are useful, most have significant drawbacks and limitations. For example, radioactive labels are dangerous and difficult to handle, fluorescent labels have limited capacity for multiplex detection because of limitations on distinguishable labels, and amplification methods can be subject to spurious signal amplification. There is a need for improved detection labels and detection techniques that can detect minute quantities of specific molecules and that can be highly multiplexed.
Analysis of protein expression and presence, such as proteome profiling or proteomics, requires sensitive detection of multiple proteins. Current methods in proteome profiling suggests that there is a shortage of tools necessary for such detection (Haynes and Yates, Proteome profiling-pitfalls and progress. Yeast 17(2):81-87 (2000)). While the techniques of chromatography and capillary electrophoresis are amenable to proteomic studies and have seen significant development efforts (see for example, Krull et al., Specific applications of capillary electrochromatography to biopolymers, including proteins, nucleic acids, peptide mapping, antibodies, and so forth. J Chromatogr A, 887:137-63 (2000), Hage, Affinity chromatography: a review of clinical applications. Clin Chem, 45(5):593-615 (1999), Hage et al., Chromatographic Immunoassays., Anal Chem, 73(07):198 A-205 A, (2001), Krull et al., Labeling reactions applicable to chromatography and electrophoresis of minute amounts of proteins. J Chromatogr B Biomed Sci Appl, 699:173-208 (1997)), the workhorse of the industry remains two dimensional electrophoresis where the two dimensions are isoelectric focusing and molecular size. Haynes and Yates point out the significant shortcomings of the technique but discuss the utility of the method in light of such shortcomings. Hayes and Yates also discuss the techniques of Isotope Coded Affinity Tags (ICAT), LC-LC-MS/MS, and stable isotope labeling techniques (Shevchenko et al., Rapid xe2x80x98de novoxe2x80x99 peptide sequencing by a combination of nanoelectrospray, isotopic labeling and a quadrupole/time-of-flight mass spectrometer. Rapid Commun Mass Spectrom 11(9):1015-1024 (1997); Oda et al., Accurate quantitation of protein expression and site-specific phosphorylation. Proc Natl Acad Sci USA 96(12):6591-6596 (1999)).
Aebersold et al. (WO 00/11208) have described labels of the composition PRG-L-A, where PRG is a protein reactive group, L is a linker (that may contain isotopically distinguishable composition), and A is an affinity moiety. Aebersold et al. describes a method where the protein reactive group is used to attach the label to a protein, an affinity capture molecule is used to capture the affinity moiety, the remaining proteins are discarded, then the affinity moiety is released and the labeled proteins are detected by mass spectrometry. The method of Aebersold et al. does not involve fragmentation or other modification of the labels or proteins.
The technique of ICAT, where cysteine residues are labeled with heavy or light tags that each contain affinity moieties, in control and tester samples, has received significant interest and holds potential for protein profiling (Gygi et al., Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nat. Biotechnol. 17(10):994-999 (1999), Griffin et al., Quantitative proteomic analysis using a MALDI quadrupole time-of-flight mass spectrometer., Anal. Chem., 73:978-986 (2001)). Gygi et al. and Griffin et al. have demonstrated relative profiling of two protein samples, where the two samples are distinguished utilizing linkers containing either eight normal hydrogen or eight heavy hydrogen (deuterium) atoms. The relative concentrations of labeled proteins are determined by ratio of peaks that are separated by the corresponding 8 amu difference in the linker molecules. Current implementations have been limited to two labels. This technique does not involve fragmentation or other modification of the labels or proteins.
Mass spectrometry has been used to detect phosphorylated proteins (DeGnore and Qin, Fragmentation of phosphopeptides in an ion trap mass spectrometer. J. Am. Soc. Mass Spectrom. 9:1175-1188 (1998); Qin and Chait, Identification and characterization of posttranslational modifications of proteins by MALDI ion trap mass spectrometry. Anal Chem, 69:4002-9 (1997); Annan et al., A multidimensional electrospray MS-based approach to phosphopeptide mapping. Anal. Chem. 73:393-404 (2001)). The methods make use of a signature mass to indicate the presence of a phosphate group, for example m/z=63 and/or m/z=79 corresponding to PO231  and PO3xe2x88x92 ions in negative ion mode, or the neutral loss of 98 Daltons from the parent ion indicates the loss of H3PO4 from the phosphorylated peptide, indicate phosphorylated Ser, Tyr, Thr. Once phosphorylated amino acids are identified, the peptide containing the modification is sequenced by standard MS/MS techniques. There is a need for a high reliability, highly multiplexed readout system for proteomics.
The status of any living organism may be defined, at any given time in its lifetime, by the complex constellation of proteins that constitute its xe2x80x9cproteome.xe2x80x9dWhile the complete status of the proteome could be defined by listing all proteins present (including modified variants) as well as their intracellular locations and concentrations, such a task is beyond the capabilities of any current single analytical method. However, attempts have been made to define the status of a cell or tissue by identifying and measuring the relative concentrations of a small subset of proteins. For example, Conrads et al., Analytical Chemistry, 72:3349-3354 (2000), have described the use of xe2x80x9cAccurate Mass Tagsxe2x80x9d (AMT) for proteome-wide protein identification. Conrads et al. show, for a simple organism, that a mass spectrometer of sufficient mass accuracy and resolution can be used to detect certain tryptic digest fragments from proteins. Once identified, the AMTs may be directly detected in samples by tryptic digest of the proteins, and high accuracy, high resolution mass spectrometry.
While the concept of Accurate Mass Tags is useful for protein discovery, as well as for generating peptide patterns in conventional biological experiments, it does not solve the problem of sensitivity that is at the heart of a truly useful diagnostic multi-protein assessment. A useful assessment consisting of AMTs will require samples containing a minimum of 2000 to 10,000 cells in order to permit reliable readout. This is so because many important cellular proteins are present at levels of only 500 to 5000 molecules per cell. If a clinically relevant protein is present in 500 copies per cell, and a precious clinical sample from a cancer patient contains only 1000 cells, the total number of proteins is 500,000, an amount that lies below the limit of detection by conventional mass spectrometry. Thus, the types of measurements proposed by Conrads et al. for the study of proteomes after identification of AMTs are not suitable for addressing important clinical problems such as the diagnosis of cancer.
Disclosed are compositions and methods for sensitive detection of one or multiple analytes. In general, the methods involve the use of special label components, referred to as reporter signals, that can be associated with, incorporated into, or otherwise linked to the analytes. Reporter signals can also be used merely in conjunction with analytes, with no significant association between the analytes and reporter signals. Compositions where reporter signals are associated with, incorporated into, or otherwise linked to the analytes are referred to as reporter signal/analyte conjugates. Such conjugates include reporter signals associated with analytes, such as a reporter signal probe hybridized to a nucleic acid sequence; reporter signals covalently coupled to analytes, such as reporter signals linked to proteins via a linking group; and reporter signals incorporated into analytes, such as fusions between a protein of interest and a peptide reporter signal.
In some embodiments, the reporter signals can be altered such that the altered forms of different reporter signals can be distinguished from each other. Reporter signal/analyte conjugates can be altered, generally through alteration of the reporter signal portion of the conjugate, such that the altered forms of different reporter signals, altered forms of different reporter signal/analyte conjugates, or both, can be distinguished from each other. Where the reporter signal or reporter signal/analyte conjugate is altered by fragmentation, any, some, or all of the fragments can be distinguished from each other, depending on the embodiment. For example, where reporter signals fragmented into two parts, either or both parts of the reporter signals can be distinguished. Where reporter signal/analyte conjugates are fragmented into two parts (with the break point in the reporter signal portion), either the reporter signal fragment, the reporter signal/analyte fragment, or both can be distinguished. In some embodiments, only one part of a fragmented reporter signal will be detected and so only this part of the reported signals need be distinguished.
In some embodiments, sets of reporter signals can be used where two or more of the reporter signals in a set have one or more common properties that allow the reporter signals having the common property to be distinguished and/or separated from other molecules lacking the common property. In other embodiments, sets of reporter signal/analyte conjugates can be used where two or more of the reporter signal/analyte conjugates in a set have one or more common properties that allow the reporter signal/analyte conjugates having the common property to be distinguished and/or separated form other molecules lacking the common property. In still other embodiments, analytes can be fragmented (prior to or following conjugation) to produce reporter signal/analyte fragment conjugates (which can be referred to as fragment conjugates). In such cases, sets of fragment conjugates can be used where two or more of the fragment conjugates in a set have one or more common properties that allow the fragment conjugates having the common property to be distinguished and/or separated from other molecules lacking the common property. It should be understood that fragmented analytes can be considered analytes in their own right. In this light, reference to fragmented analytes is made for convenience and clarity in describing certain embodiments and to allow reference to both the base analyte and the fragmented analyte.
As indicated above, reporter signals conjugated with analytes can be altered while in the conjugate and distinguished. Conjugated reporter signals can also be dissociated or separated, in whole or in part, from the conjugated analytes prior to their alteration. Where the reporter signals are dissociated (in whole or in part) from the analytes, the method can be performed such that the fact of association between the analyte and reporter signal is part of the information obtained when the reporter signal is detected. In other words, the fact that the reporter signal may be dissociated from the analyte for detection does not obscure the information that the detected reporter signal was associated with the analyte.
Reporter signals can also be in conjunction with analytes (such as in mixtures of reporter signals and analytes), where no significant physical association between the reporter signals and analytes occurs; or alone, where no analyte is present. In such cases, where reporter signals are not or are no longer associated with analytes, sets of reporter signals can be used where two or more of the reporter signals in a set have one or more common properties that allow the reporter signals having the common property to be distinguished and/or separated from other molecules lacking the common property.
Detection of the reporter signals indicates the presence of the corresponding analytes. The reporter signals preferably can have two key features. First, the reporter signals can be used in sets where all the reporter signals in the set have similar properties (for example, mass spectrometry reporter signals may have similar mass-to-charge ratios). The similar properties allow the reporter signals to be distinguished and/or separated from other molecules lacking one or more of the properties. Second, all the reporter signals in a set can be fragmented, decomposed, reacted, derivatized, or otherwise modified to distinguish the different reporter signals in the set. Preferably, mass spectrometry reporter signals are fragmented to yield fragments of similar charge but different mass.
Differential distribution of mass in the fragments of the reporter signals can be accomplished in a number of ways. For example, reporter signals of the same nominal structure (for example, peptides having the same amino acid sequence) can be made with different distributions of heavy isotopes, such as deuterium; reporter signals of the same nominal structure can be made with different distributions of modifications, such as methylation, phosphorylation, sulphation, and use of seleno-methionine for methionine; reporter signals of the same nominal composition (for example, made up of the same amino acids) can be made with different ordering of the subunits or components of the reporter signal; and reporter signals having the same nominal composition can be made with a labile or scissile bond at a different location in the reporter signal. Each of these modes can be combined with each other and/or one or more of the other modes to produce differential distribution of mass in the fragments of the reporter signals.
The reporter signals are preferably detected using mass spectrometry which allows sensitive distinctions between molecules based on their mass-to-charge ratios. The disclosed reporter signals can be used as general labels in myriad labeling and/or detection techniques. A set of isobaric reporter signals can be used for multiplex labeling and/or detection of many analytes since the reporter signal fragments can be designed to have a large range of masses, with each mass individually distinguishable upon detection.
The disclosed method has advantageous properties which can be used as a detection system in a number of fields, including antibody or protein microarrays, DNA microarrays, expression profiling, comparative genomics, immunology, diagnostic assays, and quality control.
A. Reporter Molecule Labeling
In one form of the disclosed method, referred to as reporter molecule labeling (RML), reporter signals are first associated with the analytes and then dissociated and detected. The reporter signals preferably are associated with the analytes via interaction of specific binding molecules with the analytes. The reporter signals are either directly or indirectly associated with the specific binding molecules such that interaction of the specific binding molecules with the analytes allows the reporter signals to be associated with the analytes. The method can be performed such that the fact of association between the analyte and reporter signal is part of the information obtained when the reporter signal is detected. In other words, the fact that the reporter signal may be dissociated from the analyte for detection does not obscure the information that the detected reporter signal was associated with the analyte.
B. Reporter Signal Labeling
In another form of the disclosed method, referred to as reporter signal labeling (RSL), reporter signals are used for sensitive detection of one or multiple analytes. In the method, analytes labeled with reporter signals are analyzed using the reporter signals to distinguish the labeled analytes (where the analytes are labeled with the reporter signals). Detection of the reporter signals indicates the presence of the corresponding analyte(s). The detected analyte(s) can then be analyzed using known techniques. The labels provide a unique analyte/label composition that can specifically identify the analyte(s). This is accomplished through the use of the specialized reporter signals as the labels. The labeled analyte(s) can be fragmented prior to analysis. An analyte sample to be analyzed can also be subjected to fractionation or separation to reduce the complexity of the samples. Fragmentation and fractionation can also be used together in the same assay. Such fragmentation and fractionation can simplify and extend the analysis of the analytes.
Reporter signals can be coupled or directly associated with an analyte. For example, a reporter signal can be coupled to an analyte via reactive groups, or a reporter molecule (composed of a specific binding molecule and a reporter signal) can be associated with an analyte. The reporter signals can be attached to analytes in any manner. For example, reporter signals can be covalently coupled to proteins through a sulfurxe2x80x94sulfur bond between a cysteine on the protein and a cysteine on the reporter signal. Many other chemistries and techniques for coupling compounds to analytes are known and can be used to couple reporter signals to analytes. For example, coupling can be made using thiols, epoxides, nitrites for thiols, NHS esters, isothiocyanates for amines, and alcohols for carboxylic acids. Reporter signals can be attached to analytes either directly or indirectly, for example, via a linker.
Alternatively, a reporter signal can be associated with an analyte indirectly. In this mode, a xe2x80x9ccodingxe2x80x9d molecule containing a specific binding molecule and a coding tag can be associated with the analyte (via the specific binding molecule). Alternatively, a coding tag can be coupled or directly associated with the analyte. Then a reporter signal associated with a decoding tag (such a combination is another form of reporter molecule) is associated with the coding molecule through an interaction between the coding tag and the decoding tag. An example of this interaction is hybridization where the coding and decoding tags are complementary nucleic acid sequences. The result is an indirect association of the reporter signal with the analyte. This mode has the advantage that all of the interactions of the reporter signals with the coding molecule can be made chemically and physically similar by using the same types of coding tags and decoding tags for all of the coding molecules and reporter molecules in a set.
Reporter signals can be fragmented, decomposed, reacted, derivatized, or otherwise modified, preferably in a characteristic way. This allows an analyte to which the reporter signal is attached to be identified by the correlated detection of the labeled analyte and one or more of the products of the labeled analyte following fragmentation, decomposition, reaction, derivatization, or other modification of the reporter signal (the labeled analyte is the analyte/reporter signal combination). The alteration of the reporter signal will alter the labeled analyte in a characteristic and detectable way. Together, the detection of a characteristic labeled analyte and a characteristic product of the labeled analyte can uniquely identify the analyte. In this way, using the disclosed method and materials, one or more analytes can be detected, either alone or together (for example, in a multiplex assay). Further, one or more analytes in one or more samples can be detected in a multiplex manner. Preferably, for mass spectrometry reporter signals, the reporter signals are fragmented to yield fragments of similar charge but different mass.
In preferred embodiments, reporter signals are used in sets where all the reporter signals in the set have similar properties (such as similar mass-to-charge ratios). The similar properties allow the reporter signals to be distinguished and/or separated from other molecules lacking one or more of the properties. Alternatively, or in addition, reporter signals can be used in sets such that the resulting labeled analytes will have similar properties allowing the labeled analytes to be distinguished and/or separated from other molecules lacking one or more of the properties.
Analytes can be detected using the disclosed reporter signals in a variety of ways. For example, the analyte and attached reporter signal can be detected together, one or more fragments of the analyte and the attached reporter signal(s) can be detected together, the fragments of the reporter signal can be detected, or a combination. Preferred detection involves detection of the analyte/reporter signal both before and after fragmentation of the reporter signal.
A preferred form of the disclosed method involves correlated detection of the reporter signals both before and after fragmentation of the reporter signal. This allows labeled analytes to be detected and identified via the change in labeled analyte. That is, the nature of the reporter signal detected (non-fragmented versus fragmented) identifies the analyte as labeled. Where the analytes and reporter signals are detected by mass-to-charge ratio, the change in mass-to-charge ratio between fragmented and non-fragmented samples provides the basis for comparison. Such mass-to-charge ratio detection is preferably accomplished with mass spectrometry.
As an example, an analyte in a sample can be labeled with reporter signal designed as a mass spectrometry label. The labeled analyte can be subjected to mass spectrometry. A peak corresponding to the analyte/reporter signal will be detected. Fragmentation of the reporter signal in a collision cell in the mass spectrometer would result in a shift in the peak corresponding to the loss of a portion of the attached reporter signal, the appearance of a peak corresponding to the lost fragment, or a combination of both events. Significantly, the shift observed will depend on which reporter signal is on the analyte since different reporter signals will, by design, produce fragments with different mass-to-charge ratios. The combination event of detection of the parent mass-to-charge (with no collision gas) and the mass-to-charge corresponding to the loss of the fragment from the reporter signal (with collision gas) indicates a labeled analyte. The identity of the analyte can be determined by standard mass spectrometry techniques, such as compositional analysis.
A powerful form of the disclosed method is use of analytes labeled with reporter signals to assay multiple samples (for example, time series assays or other comparative analyses). Knowledge of the temporal response of a biological system following perturbation is a very powerful process in the pursuit of understanding the system. To follow the temporal response, a sample of the system is obtained (for example, cells from a cell culture, mice initially synchronized and sacrificed) at determined times following the perturbation. Knowledge of spatial analyte profiles (for example, relative position within a tissue section) is a very powerful process in the pursuit of understanding the biological system.
In the disclosed method a series of samples can each be labeled with a different reporter signal from a set of reporter signals. Preferred reporter signals for this purpose would be those using differentially distributed mass. In particular, the use of stable isotopes is preferred to ensure that members of the set of reporter signals would behave chemically identically and yet would be distinguishable.
The labeled analytes are preferably detected using mass spectrometry which allows sensitive distinctions between molecules based on their mass-to-charge ratios. The disclosed reporter signals can be used as general labels in myriad labeling and/or detection techniques. A set of isobaric reporter signals can be used for multiplex labeling and/or detection of many analytes since the reporter signal fragments can be designed to have a large range of masses, with each mass individually distinguishable upon detection. Where the same analyte or type of analyte is labeled with a set of isobaric reporter signals (by, for example, labeling the same analyte in different samples), the set of labeled analytes that results from use of an isobaric set of reporter signals will also be isobaric. Fragmentation of the reporter signals will split the set of labeled analytes into individually detectable labeled proteins of characteristically different mass.
The disclosed method can be used in many modes. For example, the disclosed method can be used to detect a specific analyte (in a specific sample or in multiple samples) or multiple analytes (in a single sample or multiple samples). In each case, the analyte(s) to be detected can be separated either from other, unlabeled analytes or from other molecules lacking a property of the labeled analyte(s) to be detected. For example, analytes in a sample can be generally labeled with reporter signals and some analytes can be separated on the basis of some property of the analytes. For example, the separated analytes could have a certain mass-to-charge ratio (separation based on mass-to-charge ratio will select both labeled and unlabeled analytes having the selected mass-to-charge ratio). As another example, all of the labeled analytes can be distinguished and/or separated from unlabeled molecules based on a feature of the reporter signal such as an affinity tag. Where different affinity tags are used, some labeled analytes can be distinguished and/or separated from others. Reporter signal labeling allows profiling of analytes and cataloging of analytes.
In one mode of the disclosed method, multiple analytes in multiple samples are labeled where all of the analytes in a given sample are labeled with the same reporter signal. That is, the reporter signal is used as a general label of the analytes in a sample. Each sample, however, uses a different reporter signal. This allows samples as a whole to be compared with each other. By additionally separating or distinguishing different analytes in the samples, one can easily analyze many analytes in many samples in a single assay. For example, proteins in multiple samples can be labeled with reporter signals as described above, and the samples mixed together. If some or all of the various labeled proteins are separated by, for example, association of the proteins with antibodies on an array, the presence and amount of a given protein in each of the samples can be determined by identifying the reporter signals present at each array element. If the protein corresponding to a given array element was present in a particular sample, then some of the protein associated with that array element will be labeled with the reporter signal used to label that particular sample. Detection of that reporter signal will indicate this. This same relationship holds true for all of the other samples. Further, the amount of reporter signal detected can indicate the amount of a given protein in a given sample, and the simultaneous quantitation of protein in multiple samples can provide a particularly accurate comparison of the levels of the proteins in the various samples.
In one form of reporter signal labeling, referred to as reporter signal protein labeling (RSPL), reporter signals are used for sensitive detection of one or multiple proteins. In the method, proteins labeled with reporter signals are analyzed using the reporter signals to distinguish the labeled proteins. Detection of the reporter signals indicates the presence of the corresponding protein(s). The detected protein(s) can then be analyzed using known techniques. The labels provide a unique protein/label composition that can specifically identify the protein(s). This is accomplished through the use of the specialized reporter signals as the labels. The labeled protein(s) can be fragmented, such as by protease digestion, prior to analysis. A protein sample to be analyzed can also be subjected to fractionation or separation to reduce the complexity of the samples. Fragmentation and fractionation can also be used together in the same assay. Such fragmentation and fractionation can simplify and extend the analysis of the proteins.
C. Reporter Signal Calibration
In another form of the method, referred to as reporter signal calibration (RSC), a form of reporter signals referred to as reporter signal calibrators are mixed with analytes or analyte fragments, the reporter signal calibrators and the analytes or analyte fragments are altered, and the altered forms of the reporter signal calibrators and altered forms of the analytes or analyte fragments are detected. Reporter signal calibrators are useful as standards for assessing the amount of analytes present. That is, one can add a known amount of a reporter signal calibrator in order to assess the amount of analyte present comparing the amount of altered analyte or analyte fragment detected with the amount of altered reporter signal calibrator detected and calibrating these amounts with the known amount of reporter signal calibrator added (and thus the predicted amount of altered reporter signal calibrator).
In some embodiments, each analyte or analyte fragment can share one or more common properties with at least one reporter signal calibrator such that the reporter signal calibrators and analytes or analyte fragments having the common property can be distinguished and/or separated from other molecules lacking the common property.
In some embodiments, reporter signal calibrators and analytes and analyte fragments can be altered such that the altered form of an analyte or analyte fragment can be distinguished from the altered form of the reporter signal calibrator with which the analyte or analyte fragment shares a common property. In some embodiments, the altered forms of different reporter signal calibrators can be distinguished from each other. In some embodiments, the altered forms of different analytes or analyte fragments can be distinguished from each other.
In some embodiments of reporter signal calibration, the analyte or analyte fragment is not altered and so the altered reporter signal calibrators and the analytes or analyte fragments are detected. In this case, the analyte or analyte fragment can be distinguished from the altered form of the reporter signal calibrator with which the analyte or analyte fragment shares a common property.
In some embodiments the analyte or analyte fragment may be a reporter signal or a fragment of a reporter signal. In this case, the reporter signal calibrators serve as calibrators for the amount of reporter signal detected.
Reporter signal calibration is preferably used in connection with proteins and peptides (as the analytes). This form of reporter signal calibration is referred to as reporter signal protein calibration. Reporter signal protein calibration is useful, for example, for producing protein signatures of protein samples. As used herein, a protein signature is the presence, absence, amount, or presence and amount of a set of proteins or protein surrogates.
In some embodiments of reporter signal protein calibration, the presence of labile, scissile, or cleavable bonds in the proteins to be detected can be exploited. Peptides, proteins, or protein fragments (collectively referred to, for convenience, as protein fragments in the remaining description) containing such bonds can be altered by fragmentation at the bond. In this way, reporter signal calibrators having a common property (such as mass-to-charge ratio) with the protein fragments can be used and the altered forms of the reporter signal calibrators and the altered (that is, fragmented) forms of the protein fragments can be detected and distinguished. In this regard, although the protein fragments share a common property with their matching reporter signal calibrators, the altered forms of the reporter signal calibrators and altered forms of protein fragments can be distinguished (because, for example, the altered forms have different properties, such as different mass-to-charge ratios).
D. Reporter Signal Fusions
In another form of the disclosed method and compositions, referred to as reporter signal fusions (RSF), reporter signal peptides are joined with a protein or peptide of interest in a single amino acid segment. Such fusions of proteins with reporter signal peptides can be expressed from a nucleic acid molecule encoding the amino acid segment that constitutes the fusion. The fusion protein is referred to herein as a reporter signal fusion. The reporter signal peptides allow for sensitive monitoring and detection of the proteins and peptides to which they are fused, and of expression of the genes, vectors, expression constructs, and nucleic acids that encode them. In particular, the reporter signal fusions allow sensitive and multiplex detection of expression of particular proteins and peptides of interest, and/or of the genes, vectors, and expression constructs encoding the proteins and peptides of interest.
The disclosed reporter signal fusions also are useful for creating cells, cell lines, and organisms that have particular protein(s), gene(s), vector(s), and/or expression sequence(s) labeled (that is, associated with or involved in) reporter signal fusions. For example, a set of nucleic acid constructs, each encoding a reporter signal fusion with a different reporter signal peptide, can be used to uniquely label a set of cells, cell lines, and/or organisms. Processing of a sample from any of the labeled sources can result in a unique altered form of the reporter signal peptide (or the amino acid segment or an amino acid subsegment) for each of the possible sources that can be distinguished from the other altered forms. Detection of a particular altered form identifies the source from which it came.
The disclosed reporter signal fusions also can be used to xe2x80x9clabelxe2x80x9d particular pathways, regulatory cascades, and other suites of genes, proteins, vectors, and/or expressions sequences. Such labeling can be within the same cell, cell line, or organism, or across a set of cells, cell lines, or organisms. For example, nucleic acid segments encoding reporter signal fusions can be associated with endogenous expression sequences of interest, endogenous genes of interest, exogenous expression sequences of interest, exogenous genes of interest, or a combination. The exogenous constructs then are introduced into the cells or organisms of interest. The association with endogenous expression sequences or genes can be accomplished, for example, by introducing a nucleic acid molecule (encoding the reporter signal fusion) for insertion at the site of the expression sequences or gene of interest, or by creating a vector or other nucleic acid construct (containing both the endogenous expression sequences or gene and a nucleic acid segment encoding the reporter signal fusion) in vitro and introducing the construct into the cells or organisms of interest. Many other uses and modes of use are possible, a number of which are described in the illustrations below. In particular, the disclosed reporter signal fusions can be used in any context and for any purpose that green fluorescent protein and green fluorescent protein fusions are used. However, the disclosed reporter signal proteins have more uses and are more useful than green fluorescent protein at least because of the ability to multiplex the disclosed reporter signal fusions.
The reporter signal peptides can be used for sensitive detection of one or multiple proteins (that is, the proteins to which the reporter signal peptides are fused). In the method, proteins fused with reporter signal peptides are analyzed using the reporter signal peptides to distinguish the reporter signal fusions. Detection of the reporter signal peptides indicates the presence of the corresponding protein(s). The detected protein(s) can then be analyzed using known techniques. The reporter signal fusions provide a unique protein/label composition that can specifically identify the protein(s). This is accomplished through the use of the specialized reporter signal peptides as the labels.
The reporter signal fusions can be produced by expression from nucleic acid molecules encoding the fusions. Thus, the disclosed fusions generally can be designed by designing nucleic acid segments that encode amino acid segments where the amino acid segments comprise a reporter signal peptide and a protein or peptide of interest. A given nucleic acid molecule can comprise one or more nucleic acid segments. A given nucleic acid segment can encode one or more amino acid segments. A given amino acid segment can include one or more reporter signal peptides and one or more proteins or peptides of interest. The disclosed amino acid segments consist of a single, contiguous polypeptide chain. Thus, although multiple amino acid segments can be part of the same contiguous polypeptide chain, all of the components (that is, the reporter signal peptide(s) and protein(s) and peptide(s) of interest) of a given amino acid segment are part of the same contiguous polypeptide chain.
Reporter signal peptides can be fragmented, decomposed, reacted, derivatized, or otherwise modified, preferably in a characteristic way. This allows a protein to which the reporter signal peptide is fused to be identified by detection of one or more of the products of the reporter signal fusion following fragmentation, decomposition, reaction, derivatization, or other modification of the reporter signal peptide. Expression of one or more proteins in one or more samples can be detected in a multiplex manner. Preferably, for mass spectrometry reporter signals, the reporter signal peptides are fragmented to yield fragments of similar charge but different mass.
Preferably, the reporter signal peptides are fragmented to yield fragments of similar charge but different mass. This allows each reporter signal fusion (and/or each reporter signal peptide) in a set to be distinguished by the different mass-to-charge ratios of the fragments of (that is, altered forms of) the reporter signal peptides or reporter signal fusions. This is possible since the fragments of the different reporter signal peptides (or the fragments of the reporter signal fusions) can be designed to have different mass-to-charge ratios. In the disclosed method, this allows each reporter signal fusion to be distinguished by the mass-to-charge ratios of the reporter signal fusions after fragmentation of the reporter signal peptide.
Alteration of reporter signals peptides in reporter signal fusions can produce a variety of altered compositions. Any or all of these altered forms can be detected. For example, the altered form of the reporter signal peptide can be detected, the altered form of the amino acid segment (which contains the reporter signal peptide) can be detected, the altered form of a subsegment of the amino acid segment can be detected, or a combination of these can be detected. Where the reporter signal peptide is altered by fragmentation, the result generally will be a fragment of the reporter signal peptide and an altered form of the amino acid segment containing the protein or peptide of interest and a portion of the reporter signal peptide (that is, the portion not in the reporter signal peptide fragment). The protein or peptide of interest also can be fragmented. The result would be a subsegment of the amino acid segment. The amino acid subsegment would contain the reporter signal peptide and a portion of the protein or peptide of interest. When the reporter signal peptide in an amino acid subsegment is altered (which can occur before, during, or after fragmentation of the amino acid segment), the result is an altered form of the amino acid subsegment (and an altered form of the reporter signal peptide). This altered form of amino acid subsegment can be detected.
As with reporter signals generally, reporter signal peptides can be used in sets where the reporter signal peptides in a set can have one or more common properties that allow the reporter signal peptides to be separated or distinguished from molecules lacking the common property. In the case of reporter signal fusions, amino acid segments and amino acid subsegments can be used in sets where the amino acid segments and amino acid subsegments in a set can have one or more common properties that allow the amino acid segments and amino acid subsegments, respectively, to be separated or distinguished from molecules lacking the common property. In general, the component(s) of the reporter signal fusions having common properties can depend on the component(s) to be detected and/or the mode of the method being used.
Nucleic acid molecules encoding reporter signal fusions can be used in sets where the reporter signal peptides in the reporter signal fusions encoded by a set of nucleic acid molecules can have one or more common properties that allow the reporter signal peptides to be separated or distinguished from molecules lacking the common property. Similarly, nucleic acid segments (which, generally, are part of nucleic acid molecules) encoding reporter signal fusions can be used in sets where the reporter signal peptides in the reporter signal fusions encoded by a set of nucleic acid segments can have one or more common properties that allow the reporter signal peptides to be separated or distinguished from molecules lacking the common property. Other relationships between members of the sets of nucleic acid molecules, nucleic acid segments, amino acid segments, reporter signal peptides, and proteins of interest are contemplated.
Cells, cell lines, organisms, and expression of genes and proteins can be detected using the disclosed reporter signal fusions in a variety of ways. For example, the protein and attached reporter signal peptide can be detected together, one or more peptides of the protein and the attached reporter signal peptide(s) can be detected together, the fragments of the reporter signal peptide can be detected, or a combination. Preferred detection involves detection of the reporter signal fusion both before and after fragmentation of the reporter signal peptide.
A powerful form of the disclosed method is use of reporter signal fusions to assay multiple samples (for example, time series assays or other comparative analyses). Knowledge of the temporal response of a biological system following perturbation is a very powerful process in the pursuit of understanding the system. To follow the temporal response a sample of the system is obtained (for example, cells from a cell culture, mice initially synchronized and sacrificed) at determined times following the perturbation. Knowledge of spatial protein profiles (for example, relative position within a tissue section) is a very powerful process in the pursuit of understanding the biological system.
The reporter signal fusions are preferably detected using mass spectrometry which allows sensitive distinctions between molecules based on their mass-to-charge ratios. A set of isobaric reporter signal peptides or reporter signal fusions can be used for multiplex labeling and/or detection of the expression of many genes, proteins, vectors, expression constructs, cells, cell lines, and organisms since the reporter signal peptide fragments can be designed to have a large range of masses, with each mass individually distinguishable upon detection. Where the same gene, protein, vector, expression construct, cell, cell line, or organism (or the same type of gene, protein, vector, expression construct, cell, cell line, or organism) is labeled with a set of reporter signal fusions that are isobaric or that include isobaric reporter signal peptides (by, for example, xe2x80x9clabelingxe2x80x9d the same gene, protein, vector, expression construct, cell, cell line, or organism in different samples), the set of reporter signal fusions or reporter signal peptides that results will also be isobaric. Fragmentation of the reporter signal peptides will split the set of reporter signal peptides into individually detectable reporter signal fusion fragments and reporter signal peptide fragments of characteristically different mass.
A preferred form of the disclosed method involves filtering of isobaric reporter signal fusions or reporter signal peptides from other molecules based on mass-to-charge ratio, fragmentation of the reporter signal peptides to produce fragments having different masses, and detection of the different fragments based on their mass-to-charge ratios. The method is best carried out using a tandem mass spectrometer as described elsewhere herein.
It is an object of the present invention to provide a method for the multiplexed determination of presence, amount, or presence and amount of analytes.
It is an object of the present invention to provide labeled proteins such that the presence, amount, or presence and amount of the proteins can be determined.
It is another object of the present invention to provide a method for labeling proteins so as to allow the multiplexed determination of presence, amount, or presence and amount of proteins.
It is another object of the present invention to provide a method for the multiplexed determination of presence, amount, or presence and amount of proteins.
It is an object of the present invention to provide a method for detecting a mass tag signature.
It is an object of the present invention to provide a method for detecting a protein signature.
It is another object of the present invention to provide an assessment of the identity and purity of the peptides comprising a protein signature.
It is another object of the present invention to provide a method for detecting phosphopeptides, or other posttranslational protein modifications, among the peptides comprising a protein signature.
It is another object of the present invention to provide kits for generating mass tag signatures.
It is another object of the present invention to provide kits for generating protein signatures.